Immunotherapy has recently drawn attention as a fourth method following surgery, chemotherapy and radiation therapy for treating tumors. Since immunotherapy utilizes the immunity inherent to humans, it is said that the physical burden on patients are less in immunotherapy than those in other therapies. The therapeutic approaches known as immunotherapies include: cell transfer therapy in which cells such as lymphokine-activated cells, natural killer T-cells or γδT cells obtained, for example, from exogenously-induced cytotoxic T-lymphocytes (CTLs) or peripheral blood lymphocytes by expansion culture using various method are transferred; dendritic cell-transfer therapy or peptide vaccine therapy by which in vivo induction of antigen-specific CTLs is expected; Th1 cell therapy; and immune gene therapy in which genes expected to have various effects are introduced ex vivo into the above-mentioned cells to transfer them in vivo. In these immunotherapies, CD4-positive T cells and CD8-positive T cells have traditionally been known to play a critical role.
In humans with cancer, antitumor immunity is often ineffective due to the tight regulation associated with the maintenance of immune homeostasis. One of the major limitations is a process known as ‘T-cell exhaustion’, which results from chronic exposure to antigens and is characterized by the upregulation of inhibitory receptors. These inhibitory receptors serve as immune checkpoints in order to prevent uncontrolled immune reactions. Blocking of one or several of these immune checkpoints with monoclonal antibodies (mAbs) has been shown to rescue otherwise exhausted antitumor T cells, and most importantly, has been associated with objective clinical responses in cancer patients.
Optimal T cell activation requires contemporaneous signals through the T cell receptor and costimulatory molecules. CD28, the prototypical costimulatory molecule, upon interaction with its ligands B7-1 and B7-2, plays a crucial role in initial T cell priming (see, e.g., Sharpe et al., (2002) Nat. Rev. Immunol. 2:203-209). CD28-mediated T cell expansion is opposed by another B7-1,2 counter receptor, cytotoxic T lymphocyte associated antigen 4 (CTLA-4), which attenuates the proliferation of recently activated T cells (see, e.g., Krummel et al., (1996) J. Exp. Med. 183:2533-2540; Leach et al., (1996) Science 271:1734-1736).
The identification and characterization of additional CD28 and B7 family members PD-1 (programmed death-1), PD-L1 (programmed death ligand-1 or B7-H1), and PD-L2 (B7-DC) has added further complexity to the process of T-cell activation and peripheral tolerance in humans. Similar to the B7-1 and B7-2/CTLA-4 interaction, PD-1 interactions with PD-L1 and PD-L2 downregulate central and peripheral immune responses (see, e.g., Fife et al., (2008) Immunol. Rev. 224:166-82). Accordingly, antibody-based blockade of PD-1, like CTLA-4, is also being explored in human clinical trials for the treatment of cancer (see, e.g., Berger et al. (2008) Clin. Cancer Res. 14:3044-3051). Nevertheless, as with CTLA-4, improved therapies are still needed.
In vivo electroporation is a gene delivery technique that has been used successfully for efficient delivery of plasmid DNA to many different tissues. Studies have reported the administration of in vivo electroporation for delivery of plasmid DNA to B16 melanomas and other tumor tissues. Systemic and local expression of a gene or cDNA encoded by a plasmid can be obtained with administration of in vivo electroporation. Use of in vivo electroporation enhances plasmid DNA uptake in tumor tissue, resulting in expression within the tumor, and delivers plasmids to muscle tissue, resulting in systemic expression of certain immunomodulatory molecules, such as cytokines.
It has been shown that electroporation can be used to transfect cells in vivo with plasmid DNA. Recent studies have shown that electroporation is capable of enhancing delivery of plasmid DNA as an antitumor agent. Electroporation has been administered for treatment of hepatocellular carcinomas, adenocarcinoma, breast tumors, squamous cell carcinoma and B16.F10 melanoma in rodent models. The B16.F10 murine melanoma model has been used extensively for testing potential immunotherapy protocols for the delivery of an immunostimulatory molecule including cytokines either as recombinant protein or by gene therapy.
Various protocols known in the art can be utilized for the delivery of plasmid encoding a checkpoint inhibitor utilizing in viva electroporation for the treatment of cancer. The protocols known in the art describe in vivo electroporation mediated cytokine based gene therapy, both intratumor and intramuscular, utilizing low-voltage and long-pulse currents.
Accordingly, what is needed in the art is a combination therapy of intratumoral immunostimulatory cytokine therapy and a checkpoint inhibitor, co-delivered encoded on a plasmid and delivered via electroporation to the tumor; or via systemic delivery of the checkpoint inhibitor protein therapeutic, either concurrently with or subsequent to, the intratumoral immunostimulatory cytokine therapy, that will provide substantially improved results in the regression of cancer tumors, such as melanoma, while also substantially improving the long-term survival rates.